Isolation of cyclodextrin glucanotransferase preparations of different purities

Purification of cyclodextrin glucanotransferase (EC 2.4.1.19) from the culture liquid of Bacillus megaterium was carried out by the use of three purification procedures: (a) reverse osmosis and organic solvents precipitation, (b) ultrafiltration and gel-filtration chromatography, and (c) ultrafiltration, adsorption on starch, ion exchange chromatography and gel-filtration chromatography. Best precipitation was achieved by i-propanol - 85.92% of initial enzyme activity was preserved with purification of 1.47-fold. PS-20000 appeared to be the most suitable membrane for ultrafiltration. After 8.3-fold concentration by volume, the enzyme was purified 2.46-fold with a yield of 95.30%. As a result of the two-step purification procedure (ultrafiltration with membrane PS-20000 and gel-filtration chromatography) the enzyme was purified 36.2-fold with 95.45% of enzyme activity [measured in micrograms of cyclodextrins formed in 1 ml in 1 min under the assay conditions(U)] preserved. A highly purified enzyme preparation (purification of 74.56-fold and specific activity of 60.39 U/mg protein) was obtained by the third purification procedure. The purified enzyme preparation showed a single protein band on SDS-PAGE, 10% polyacrylamide gel.     

Alteration of product specificity of cyclodextrin glucanotransferase from Thermococcus sp. B1001 by site-directed mutagenesis

Cyclodextrin glucanotransferase (CGTase) from the hyperthermophilic archaeon Thermococcus sp. B1001 catalyzed the production predominantly of alpha-cyclodextrin (CD) from starch (Tachibana, Y. et al., Appl. Environ. Microbiol., 65, 1991-1997, 1999). The CGTase gene (cgtA) from this strain was cloned and sequenced. It was composed of 2217 nucleotides, and encoded a protein (739 amino acids) with a molecular mass of 83,240 Da. Recombinant CgtA expressed in Escherichia coli also catalyzed the production predominantly of alpha-CD from starch, as did native CgtA from strain B1001. Based on a substrate binding model of Bacillus circulans no. 8 CGTase, Tyr100, Trp191 and Tyr267 were specified to locate the spiral amylose and to minimize the size of the CD by saccharide aromatics interaction. In order to determine the critical residue for catalyzing production predominantly of alpha-CD, site-directed mutations were introduced in CgtA (Y100W, Tyr100-->Trp; W191Y, Trp191-->Tyr; W191F, Trp191-->Phe; Y267W, Tyr267-->Trp; Y267F, Tyr267-->Phe). Analysis of the reaction products by HPLC revealed that the mutant enzyme Y267W produced more beta- and gamma-CD than the wild-type enzyme. However, the other mutants still produced high levels of alpha-CD, suggesting that Tyr267 plays a critical role in alpha-CD production catalyzed by B1001 CGTase.     

重组大肠杆菌摇瓶发酵产环糊精葡基转移酶条件的优化

对一株表达环糊精葡基转移酶基因工程菌E.coli BL21(DE3)产环糊精葡基转移酶的发酵条件进行了研究.利用单因素实验和正交实验获得该菌株产环糊精葡基转移酶的最佳条件为:种子培养时间14h,接种量3%,培养温度37℃,培养基初始pH8.0,发酵培养基的组成为玉米粉1.5%、玉米浆6%、蛋白胨1.0%,500mL三角瓶装液量为50mL.210r/min 振荡培养,其发酵液酶活可达5010U/mL.     

利用重组枯草芽孢杆菌L-氨基酸连接酶合成丙谷二肽

利用大肠杆菌Escherichia coli BL21(DE 3)克隆并表达来源于枯草芽孢杆菌Bacillus subtilis ATCC 15245的L-氨基酸连接酶(L-amino acid ligase,Lal)基因,采用Ni-NAT亲和层析法分离纯化得到重组的Lal,并以L-丙氨酸和L-谷氨酰胺为底物制备L-丙氨酰-L-谷氨酰胺(丙谷二肽).Lal属于ATP依赖酶,研究结果表明其最适反应温度和pH值分别为37℃和9.0,连续催化反应14 h可催化等摩尔的底物氨基酸(30 mmol/L)生成22.4 mmol/L的丙谷二肽,最高摩尔转化率可达74.7％.成功克隆表达了枯草芽孢杆菌的L-氨基酸连接酶,并将其应用于丙谷二肽的酶法合成,研究结果可为丙谷二肽的生物制造奠定理论基础.     

Enzymatic synthesis of ethyl hexanoate by transesterification

Summary Enzymatic synthesis of ethyl hexanoate by means of an acyl transfer reaction has been studied by using an immobilized Rhizomucor miehei lipase (RML). The effect of reaction parameters on ester synthesis has been investigated. Rhizomucor miehei lipase showed more specificity than other lipases when ethyl hexanoate was synthesized in n‐hexane. Maximum ester synthesis was obtained by using a 0.5 m substrate concentration (equimolar ratio). Temperatures in the range of 45–55 °C were found to be optimum and at higher temperatures (>60 °C) deactivation of enzyme was observed. Higher molar concentrations of hexanoic acid inhibited RML, but no inhibitory effect of ethyl caprate, even at higher molar concentrations, was observed. Apparent kinetic parameters have been determined. The values are as follows: K_M (ester), 0.0135 m ; K_M (acid), 0.08466; K_i (ester), 3.07 m ; K_i (acid), 0.550 m ; V_max, 1.861 µmol min^?1 mg^?1 enzyme.     

生物柴油的酶促合成研究

对固定化脂肪酶催化大豆油甲酯化反应生产生物柴油的工艺进行了研究.考察了反应条件如油醇摩尔比、甲醇的流加、温度、有机溶媒用量以及底物预处理对生物柴油产率的影响,并对产物进行了分析.结果表明,当采用乳化处理后的大豆油为底物,正己烷作为有机溶媒,固定化脂肪酶用量为原料油质量的10%,反应温度为40℃,每隔5 h流加甲醇1次,每次按油醇摩尔比1:1流加,共流加3次时,反应的转化率最高可达到94%.     

Enzymatic chemoselective synthesis of secondary-amide surfactant from N-methylethanol amine

Efficient selective synthesis of the secondary amide surfactant N-methyl lauroylethanolamide from methyl laurate and N-methylethanol amine by carrier-fixed Chirazyme L-2 (Candida antarctica) using a kinetic strategy has been demonstrated. When different solvents were screened for product yields using Chirazyme L-2, acetonitrile was found to be optimal. The rate of the reaction increased sharply by increasing the molar ratio of the reactants and the reaction temperature. When the reaction was performed at 50 degrees C for 36 h with 50 mmol ester and 100 mmol amine, the product was obtained in a 97.1% yield. With 50 mmol ester and 150 mmol amine, the highest yield (97.3%) was obtained after 16 h of incubation at 50 degrees C. It took only 5 h to get a yield of 95.8% at 60 degrees C using 50 mmol ester and 200 mmol amine. The enzyme activity in the amidation reaction mixture did not decrease notably even after six uses.     

酶法合成右旋糖酐的研究

利用肠膜状明串珠菌(Leuconstocmesenteriodes)右旋糖酐蔗糖酶催化蔗糖合成右旋糖酐.分别探讨了不同蔗糖浓度、加酶量、反应温度、pH值和反应时间等因素对蔗糖转化率的影响,从中选取影响较显著的3个因素如蔗糖浓度、反应温度、pH值,采用均匀设计试验方法对酶促反应条件进一步优化,确定酶促反应最佳工艺参数.实验结果表明:酶法合成右旋糖酐的最佳条件为:蔗糖浓度50g/L,加酶量为0.214U,反应温度12℃,pH值为7.5,反应时间为30h,蔗糖转化率约为31%.     

脂肪酸单甘油酯的酶催化合成及其表征

以癸酸甲酯、月桂酸甲酯、肉豆蔻酸甲酯、棕榈酸甲酯和硬脂酸甲酯为原料,与甘油进行脂肪酶催化酯交换反应制备脂肪酸单甘油酯,产物为脂肪酸单甘油酯和少量脂肪酸双甘油酯的混合物,其中脂肪酸单甘油酯的质量分数均在91%以上,最高可达95.9%。随着碳链长度的增加,脂肪酸甲酯的转化率降低,脂肪酸单甘油酯的质量分数也降低。采用气相色谱-质谱联机、核磁共振氢谱和红外光谱,对柱层析分离后的脂肪酸单甘油酯进行了结构表征。     

正己烷体系中酶催化菜籽油制备乙酯生物柴油

研究了脂肪酶催化菜籽油乙醇解反应的几个主要影响因素,即醇油摩尔比、催化剂用量、反应温度和反应时间,并研究了正己烷对反应的影响。得出脂肪酶催化菜籽油乙醇解制备生物柴油的最佳反应条件为:醇油摩尔比5∶1,催化剂用量10%,反应温度40℃,反应时间36 h、正己烷用量15%。在最佳反应条件下,1次加入乙醇时转化率为86%;采用3次加醇能很好改善催化环境,转化率达到93%。利用气相色谱分析了产物中脂肪酸乙酯的组成,结果表明,产物中主要是十八碳酸乙酯。     

酶促反应合成蔗糖棕榈酸酯的研究

讨论了以Novozym 435脂肪酶为催化剂,以蔗糖和棕榈酸为底物的糖酯合成反应及其影响因素.考察了在丙二醇和正己烷存在下,底物酸糖摩尔比、含水量、温度、脂肪酶浓度、时间以及摇床速度对转化率的影响.实验结果表明,底物棕榈酸与蔗糖的摩尔比为1.2∶1.0、含水量为4%,反应温度为65℃、脂肪酶浓度为棕榈酸质量的4%、反应时间为4.5 h、摇床速度为200 r/min时,棕榈酸的转化率可达到86.94%.     

响应面分析法优化柠檬酸甘油酯的合成工艺

对有机相脂肪酶催化合成柠檬酸甘油酯的工艺条件进行了优化.在单因素试验的基础上,根据Box-Benhnken试验设计原理,采用三因素三水平的响应面分析法,研究了催化剂用量、反应温度和反应时间及其交互作用对柠檬酸转化率的影响.确定最佳工艺参数为:在无水叔丁醇中,脂肪酶Novozym 435用量9.02%,54.18 ℃反应47.5 h.最佳条件下柠檬酸转化率可达77.37%.反应产物经薄层色谱分离和电喷雾质谱分析,主要为柠檬酸单脂肪酸甘油酯和单柠檬酸甘油酯.     

Enzymatic synthesis of galacto-oligosaccharides and other lactose derivatives (hetero-oligosaccharides) from lactose

Non-digestible oligosaccharides are applied as functional food ingredients to replace sucrose and to exploit specific biological functions, particularly low cariogenicity, low caloric content, prebiotic activity, and their ability to prevent adhesion of pathogens and toxins to eukaryotic cells. Oligosaccharides derived through enzymatic synthesis from lactose, i.e., galacto-oligosaccharides, lactulose and lactosucrose, account for a major part of the annual oligosaccharide production. Enzymatic production of oligosaccharides employs lactose as galactosyl-donor to transfer the galactosyl-moiety of lactose to suitable acceptor carbohydrates through the activity of β-galactosidases, or employs lactose as a galactosyl-, glucosyl- or fructosyl-acceptor through the activity of β-galactosidases, glucansucrases and fructansucrases. This communication provides an overview on the structural diversity of galacto-oligosaccharides and hetero-oligosaccharides that are produced by enzymatic conversion of lactose, and reviews the strategies used to optimize enzymatic transglycosylation with lactose as glycosyl donor or glycosyl acceptor.     

酶法合成EPA/DHA型卵磷脂

蛋黄卵磷脂和精制深海鱼油在正己烷介质中,lipolase固定化脂肪酶的作用下,通过发生酯交换反应,获得含有ω-3型多不饱和脂肪酸EPA(二十碳五烯酸)和DHA(二十二碳六烯酸)的卵磷脂.研究表明,在正已烷介质(含微量水,V水:V正=1:1000)中,蛋黄卵磷脂和鱼油底物浓度比为1:4,脂肪酶酶活为6.9 U/mL,在45℃恒温水浴中磁力搅拌反应12 h,用气相色谱分析,得到的卵磷脂中EPA和DHA的含量分别为1.40%和5.43%,总含量为6.83%.     

Enzymatic synthesis of Mycoplasma fermentans specific glycoglycerophospholipid from 1,2-dipalmitoylglycerol

A gene, mf3, encoding glycosyltransferase in glycoglycerophospholipid (GGPL; GGPL-I and GGPL-III) biosynthesis in Mycoplasma fermentans PG18 was identified by genomic analysis, cloned, modified codon usage, and expressed in Escherichia coli. The mf3 gene consists of an open reading frame of 1221 bp encoding 406 amino acids. The mf3 gene product, Mf3, has 27% amino acid homology with glycosyltransferase of Borrelia burgdorferi but no homology to genes of other Mycoplasma species in the GenBank database. The reaction product of Mf3 using 1,2-dipalmitoilglycerol and UDP-glucose as substrates showed a specific sodium adducted ion at m/z 753, which corresponded to glucopyranosyl dipalmitoilglycerol as determined by MALDI-TOF mass spectrometry. Furthermore, in the reaction product by Mf3 and Mf1 which was a cholinephosphotransferase and previously cloned from M. fermentans PG18, an ion at m/z 896 corresponding to GGPL-I was detected by mass spectrometry. The product ions of choline, phosphocholine, and hexose-bound phosphocholine were detected by tandem MS analysis of protonated molecules at m/z 896. From these results, mf3 was identified as a glycosyltransferase. It was suggested that glucose transfer and phosphocholine transfer to 1,2-dipalmitoylglycerol are involved in the GGPL biosynthesis pathway of M. fermentans PG18.     

Enzymatic synthesis of kojioligosaccharides using kojibiose phosphorylase

We have attempted to synthesize kojioligosaccharides (oligosaccharides having the alpha-1,2 glycosidic linkage at the nonreducing end) using two methods. In the first, mixtures of various proportions of glucose and beta-D-glucose-1-phosphate (beta-G1P) were allowed to react in the presence of kojibiose phosphorylase (KPase). In the second, maltose was allowed to react with KPase and maltose phosphorylase (MPase) simultaneously. In the former method, kojioligosaccharides having only the alpha-1,2 glucosidic linkage were synthesized and the average degree of polymerization (D.P.) of oligosaccharides increased with decreasing proportions of glucose. In the second method, kojioligosaccharides were obtained at approximately 70% yields under optimum conditions. 4-alpha-D-Kojibiosyl-glucose, kojitriose and kojitetraose, the principal kojioligosaccharides synthesized, were not hydrolyzed by salivary amylase, artificial gastric juice, pancreatic amylase, or small intestinal enzymes.     

硬脂酸淀粉酯的酶促合成及其乳化稳定性

硬脂酸淀粉酯是通过淀粉酯化而得到的乳化剂,它是由淀粉及其衍生物与硬脂酸、硬脂酸甲酯、硬脂酸酰氯或硬脂酸酸酐反应得到的酯化产品.硬脂酸淀粉酯由于疏水性有机碳链的引入,淀粉的疏水性增加,使之具备了亲油和亲水的双亲性质,可广泛应用于日用化学、食品、纺织化工,生物降解材料、医药等工业.目前合成硬脂酸淀粉酯所用方法主要是水媒法、溶剂法、熔融法等.本实验采用脂肪酶LipozymeTL IM为催化剂,马铃薯淀粉与硬脂酸为反应底物、焦磷酸钠为辅助剂,合成硬脂酸淀粉酯.在脂肪酶添加量为淀粉质量10%的条件下,考察了底物比,辅助剂用量,反应时间,反应温度对酯化效果的影响,确定了酶法合成硬脂酸淀粉酯的最佳条件:淀粉6.0g,硬脂酸20.0g,焦磷酸钠1.5g,反应时间42h,反应温度65℃,制得取代度为0.036的淀粉酯产品.所得淀粉酯具有良好的乳化能力.     

辛烯基琥珀酸淀粉酯的酶促合成及性能分析

以马铃薯原淀粉为原料,采用单因素和正交试验方法研究酶法合成辛烯基琥珀酸淀粉酯,从脂肪酶用量、底物比、反应温度、反应时间四个方面研究辛烯基琥珀酸淀粉酯的最佳制备工艺,其结果为:脂肪酶添加量22g/kg,辛烯基琥珀酸酐与淀粉质量比2∶1,反应时间48h,反应温度60℃。采用最佳工艺得到产品的取代度为0.042。利用红外、扫描电镜、淀粉-碘复合物吸收谱对所得到的辛烯基琥珀酸淀粉酯的结构进行分析。红外分析结果显示,淀粉酯在1728.13cm-1处出现了羰基的特征吸收峰;扫描电镜和淀粉-碘复合物紫外吸收光谱分析显示,酶法改性仅对淀粉颗粒表面造成了少数损伤,酯化反应主要发生在淀粉的无定形区,其晶体结构并未发生改变;同时其乳化能力和乳化稳定性的应用实验得到了较好的测试效果。毒理学实验证明酶法产品安全性可靠,完全可应用于食品加工制品中。     

Enzymatic synthesis of novel oligosaccharides from L-sorbose, maltose, and sucrose using kojibiose phosphorylase

Glucosyl-L-sorbose, -maltose, and -sucrose were synthesized using kojibiose phosphorylase (KPase) from Thermoanaerobacter brockii ATCC35047 with beta-D-glucose-1-phosphate (beta-G1P) as a glucosyl donor. One disaccharide and two trisaccharides thus synthesized were isolated by Toyopearl HW-40S column chromatography. The results of KPase digestion, methylation analysis, and 13C-NMR studies indicated that these oligosaccharides were alpha-D-glucopyranosyl-(1-->5)-alpha-L-sorbopyranose, alpha-D-glucopyranosyl-(1-->2)-alpha-D-glucopyranosyl-(1-->4)-D-glucopyranose (4-alpha-D-kojibiosyl-glucose), and alpha-D-glucopyranosyl-(1-->2)-alpha-D-glucopyranosyl-(1-->2)-beta-D-fructofuranoside, which are all novel oligosaccharides. Glucosyl-L-sorbose was partially hydrolyzed to glucose and L-sorbose by alpha-glucosidases, while glucosyl-sucrose and glucosyl-maltose were not hydrolyzed by glucoamylase, alpha-glucosidases, or CGTase.     

酶法提取芋艿蛋白质工艺

探讨木瓜蛋白酶对提取芋艿蛋白质的影响,获得最佳提取工艺。以木瓜蛋白酶为酶试剂提取芋艿蛋白质,以考马斯亮蓝法为蛋白质含量测定方法,采用单因素实验分别研究提取温度、酶量、pH值、提取时间对芋艿蛋白质提取率的影响,再通过正交试验对提取条件进行优化筛选,结果表明:提取温度是影响提取率的关键因素,其次分别是pH值、酶量,影响最小的因素是提取时间。此次研究得出的最佳工艺参数组合为:提取温度55℃,酶量3mg/g、pH值6.5、提取时间1h,在此条件下蛋白质提取率为10.20%。